Impact of melatonin administration on sperm quality, steroid hormone levels, and testicular blood flow parameters in small ruminants: A meta-analysis

Background and Aim The impact of exogenous melatonin on the sperm quality of small ruminants is controversial. Therefore, this study aimed to synthesize previous findings on the influence of melatonin injection on sperm quality, steroid hormones, and testicular blood flow in small ruminants. Materials and Methods Thirty studies were analyzed by computing the raw mean difference (RMD) as the effect size between the control and melatonin treatment groups, using the inverse of the variance for the random-effect model of the method of moments by DerSimonian and Laird. We assessed heterogeneity among studies using Q test. I2 statistic was used to classify the observed heterogeneity. We used Egger’s regression method to indicate publication bias. Results Melatonin injection (p < 0.05) affected sperm concentration (RMD = 0.42 × 109/mL), morphology (RMD = 2.82%), viability (RMD = 2.83%), acrosome integrity (RMD = 4.26%), and DNA integrity (RMD = 1.09%). Total motility (RMD = 5.62%), progressive motility (RMD = 7.90%), acrosome integrity (RMD = 8.68%), and DNA integrity (RMD = 2.01%) of post-thawed semen in the melatonin-treated group were also increased (p < 0.05). Similarly, treatment with melatonin (p < 0.05) enhanced total motility (RMD = 5.78%), progressive motility (RMD = 5.28%), curvilinear velocity (RMD = 4.09 μm/s), straight-line velocity (RMD = 5.61 μm/s), and average path velocity (RMD = 4.94 μm/s). Testosterone (RMD = 1.02 ng/mL) and estradiol 17-ß levels (RMD = 0.84 pg/mL) were elevated (p < 0.05) in the melatonin-injected group. Melatonin implantation ameliorated testicular blood flow, as indicated by a significant reduction (p < 0.05) in the resistive index (RMD = 0.11) and pulsatility index (RMD = –0.15). Conclusion Melatonin administration can increase the reproductive performance of small male ruminants.


Introduction
Reproductive inefficiency is a major threat to the sustainability of small ruminants, causing significant economic losses [1].The reproductive efficacy of male animals, especially small ruminants, is equally important as that of female animals [2].The quality of sperm accounts for reproductive success [3].It contributes to 50% of the flock's performance [4].Effective management of small male ruminants before and during the breeding season is essential to reduce suboptimal reproductive performance and increase the profitability and sustainability of sheep production [5,6].However, only 70%-75% of rams exhibit peak reproductive performance at the beginning of the breeding season [7].Therefore, it is necessary to devise a proper strategy to enhance the reproductive traits of small male ruminants.
Melatonin, a neurohormone secreted by the pineal gland, is a critical regulator of various physiological processes, including the regulation of circadian rhythms, antioxidant defenses, and immune modulation [8].Melatonin administration has been intensively investigated to modulate reproductive cycles in small male ruminants.Melatonin injection has beneficial effects on testosterone production, sperm, and quality of post-thawed semen in sheep and goats during Copyright: Budiyanto, et al.Open Access.This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
both breeding and non-breeding seasons .Melatonin administration also mitigates sperm abnormalities in heat-stressed rams [31].However, contradictory results have also been reported.Melatonin implantation in the ram does not correlate with sperm production or concentration [32].Similarly, sperm quality and testosterone levels do not improve in melatonin-treated rams during the breeding season [33].Furthermore, the administration of melatonin during breeding and non-breeding seasons has no impact on sperm and post-thawed semen quality, including motility and morphology in rams [34][35][36][37].In addition, testosterone levels do not differ between the untreated and melatonin-treated rams during light challenges [38].These contradictory findings necessitate a comprehensive statistical assessment to determine the influence of melatonin administration on sperm quality in small ruminants.
Meta-analysis is a statistical technique used to synthesize the results of previous studies to produce a robust quantitative conclusion [39].It provides an unbiased and objective synthesis [40,41].To the best of our knowledge, no meta-analysis has been conducted on the relationship between melatonin implantation and sperm quality in small ruminants.Therefore, this meta-analysis synthesized the results of previous studies on the influence of melatonin injection on reproduction traits and sperm quality in small ruminants.

Ethical approval
Ethical approval was not necessary for this study.The preferred reporting items for systematic review and meta-analyses (PRISMA) protocols were applied in this meta-analysis, as shown in Figure -1.

Study period and location
The meta-analysis study was conducted from August to December 2023 at Faculty of Veterinary Medicine, Gadjah Mada University, Indonesia, and National Research and Innovation Agency, Indonesia.

Search strategy
Comprehensive studies that assessed the impact of melatonin implantation on reproductive traits in small male ruminants were identified using the Science Direct, Wiley Online Library, PubMed, and Scopus databases.The search used the following keywords: "Melatonin", "Sperm", "Semen", "Ram", "Sheep", "Buck", and "Goat" which are connected through search queries such as "AND" and "OR", respectively.

Inclusion and exclusion criteria
After erasing the duplication, the identified studies were excluded if they were (1) in vitro studies, (2) review studies, (3) no full-text studies, (4) no small-ruminant studies, and (5) no melatonin implantation studies.Furthermore, the criteria for selected studies were as follows: (1) The control (non-treatment) group was available; (2) measures of variance (e.g., standard deviations [SD], standard errors [SE], or confidence intervals [CI]) were provided; and (3) the influence of melatonin injection on sperm quality, postthawed semen quality, testicular blood flow, and plasma steroid hormones in small ruminants was studied.

Extraction
The included studies are presented in Table-1  as follows: First author's name, year, location, species, season, dose, and research duration.The graphical data were extracted using WebPlotDigitizer (Automeris LLC, CA, USA) [42].SD was calculated using the following formula: (1) SD = SE√N, where N is the repetition number; and (2) SD = √N × (upper CI-lower CI)/3.92,where 3.92 is the SE of the 95% CI and replaced with the t-distribution value if the sample was <60 in publications with no report of SD [43].

Statistical analysis
Meta-analysis and meta-regression were performed using the "metafor" package (Free Software Foundation, Inc., MA, USA) [44] and R software (R Foundation, Vienna, Austria) [45].The influence of melatonin injection on reproductive traits and steroid hormones in small male ruminants was synthesized by computing the raw mean differences (RMDs) between untreated (control) and treated (melatonin injection) means, using the inverse of the variance for the random-effect model of the method of moments by DerSimonian and Laird [46].RMD was selected to measure the findings in the original units [47].

Subgroup analysis
Meta-regression was performed if the following criteria were fulfilled: (1) Heterogeneity was significant (p ≤ 0.10 or I 2 > 50%), (2) there was no publication bias (p-value of Egger's test >0.05), and (3) the number of comparisons was >10 [47].A categorical covariate was the type of season (breeding and non-breeding).In addition, days post-treatment (days) and doses of melatonin (mg) were employed as continuous covariates.We applied the method of moments proposed by DerSimonian and Laird [46] to perform meta-regression.Subgroup analysis was implemented to evaluate RMD in the presence of significant results (p ≤ 0.05) within categorical or continuous covariates.The doses of melatonin were 18, 36, 54, and more than 54 mg.Furthermore, the days post-injection were sub-grouped into 1-31, 31-60, and >60 days post-injection.

Publication bias and meta-regression analysis
Heterogeneity was significant (p < 0.05) for all parameters of sperm quality, post-thawed semen quality, sperm motility, reproductive steroid hormones, and testicular blood flow parameters (Tables -2-6).On the other hand, publication bias was not found (p > 0.05) on sperm normal morphology, sperm total motility, sperm viability, acrosome integrity, VCL, VSL, VAP, PSV, RI, PI, post-thawed progressive motility, postthawed acrosome integrity, and post-thawed DNA integrity.Meta-regression was performed when the effect size and heterogeneity were significant and publication bias was not present.

Discussion
Melatonin modulates the release of gonadotropin-releasing hormone [51], a key regulator of reproductive physiology.It is actively transported into the testes [52], modulating various cellular processes involved in spermatogenesis and steroidogenesis [53,54].Melatonin administration enhances spermatogenesis [55].
Our meta-analysis revealed that treatment with melatonin enhances sperm quality in small ruminants, including viability, concentration, normal morphology, acrosome, and DNA integrity.These findings are similar to those reported by Abbas et al. [9], who reported that injection of melatonin increases the concentration, normal morphology, acrosome, and DNA integrity of sperm in rams.Moreover, motility, acrosome, and DNA integrity in the post-thawed semen of melatonin-treated small ruminants were also increased.Shahat et al. [30] discovered that injection of melatonin improves post-thawed motility, acrosome, and DNA integrity of rams.
Blood circulation is crucial, especially for testicular function [56].Enhanced testicular blood flow increases the supply of oxygen and nutrients to the testes [57].Melatonin enhances testicular blood flow [26] and is correlated with heightened sperm quality in rams [31].Resistance (RI) and perfusion (PI) indices are key indicators extensively used for evaluating testicular blood flow in various animals [58][59][60][61].Reduced RI and PI values indicated elevated testicular blood flow [62].This study also found that elevated sperm quality is linked to low RI and PI values.Therefore, the increase in sperm and post-thawed semen quality in melatonin-treated small ruminants can be attributed to the enhancement of testicular blood supply.
This study showed that melatonin implantation improves sperm motility and velocity of small ruminants.Egerszegi et al. [13] and Casao et al. [10] also found that injection of melatonin increases sperm motility and ram velocity in the out-of-season period.Moreover, Shahat et al. [31] found that sperm motility and velocity of melatonin-treated rams subjected to mild heat-stressed challenges increased during breeding seasons.
RI and PI are negatively correlated with the progressive motility of sperm [62].Decreased arterial blood flow to the testes impairs mitochondrial energy processes, preventing spermatogenesis.This dysfunction of the energetic pathway reduces sperm motility [63].Our results revealed that sperm kinetic parameters increased concomitantly with the enhancement of testicular blood flow.Thus, we suggest that melatonin modulates testicular blood flow, resulting in increased mitochondrial energy production and enhanced sperm motility.
In this study, we found that testosterone and estradiol-17β levels are elevated in melatonin-treated small ruminants.These findings are consistent with those of previous studies where testosterone levels were elevated in melatonin-treated rams and bucks [16,31].Melatonin affects testosterone production through the anterior pituitary gland and ameliorates Leydig cell function to promote testosterone secretion in sheep [64].Samir et al. [26] found that melatonin elevates testosterone production in Shiba bucks through the hypothalamus-pituitary axis.Moreover, Casao et al. [10] discovered that the enhancement of antioxidant enzymes induces the elevation of testosterone and estradiol-17β levels in melatonin-treated rams.
Furthermore, our study demonstrated that melatonin implantation increases PSV and decreases RI and PI, indicating enhanced testicular blood supply.RI and PI levels are regulated by plasma estradiol-17β [65].Bollwein et al. [66] found that estradiol-17β levels, but not testosterone levels, control testicular blood flow in stallions.Moreover, Salama et al. [67] discovered that enhanced testicular blood flow in melatonin-treated canines is triggered by elevation of estradiol-17β levels.Similarly, El-Shafoly et al. [15] reported that melatonin-injected rams show a reduction in RI and PI levels accompanied by an increase in estradiol-17β levels.Interestingly, our study also demonstrated a correlation between decreased RI and PI and enhancement of estradiol-17β levels.Therefore, we suggest  that melatonin administration improves testicular blood flow in small ruminants by modulating estradiol-17β levels.

Conclusion
These results indicate that melatonin administration can improve sperm quality in small ruminants.The best outcomes for acrosome integrity, VCL, postthawed progressive motility, and post-thawed DNA integrity were achieved with 36 mg melatonin injection during the breeding season.As evidenced by a reduction in RI and PI, optimal results for testicular blood supply were attained with 36 mg melatonin implantation during the breeding season within 1-60 days post-injection.Moreover, melatonin administration was associated with superior results in normal morphology, motility, VSL, and VAP during the breeding season.

Figure- 3 :
Figure-3: Subgroup analysis of the season type of effect of melatonin administration on (a) resistive index, (b) pulsatility index, and (c) sperm viability, %.

Table - 2
: Effect of implanted melatonin on sperm quality of small-ruminants.

Table - 3
: Post-thawed semen quality between melatonin-treated and control small-ruminants.Comparison results of sperm kinetic parameters in melatonin-treated and control small-ruminants.Effect of exogenous melatonin on reproductive steroid hormones in male small-ruminants.

Table - 7
: Meta-regression comparing the association between covariates and measured outcomes.

Table - 8
: Subgroup analysis of season type of the effect of melatonin injection on sperm quality, sperm motility, and testicular blood flow parameters.RMD=Raw mean difference, CI=Confidence of interval, ns=Non-significant, PI=Pulsatility index, VCL=Curvilinear velocity, VSL=Straight-line velocity, VAP=Average path velocity